Enzyme preparation comprising a modified enzyme

ABSTRACT

An enzyme preparation comprising a modified enzyme selected from the group consisting of an amylase, lipase, oxidorcductase, pectinace or hemicellulase, the modified enzyme having an improved performance due to an alkaline pI and/or increased surface activity obtained by chemical modification or amino acid substitution, is useful e.g., in detergents, in baking flour, in animal feed, in the manufacture of cellulosic fabrics and for the treatment of lignocellulosic fibers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/DK94/00368 filed Oct. 4, 1994, which is incorporated herein byreference.

FIELD OF INVENTION

The present invention relates to an enzyme preparation comprising amodified enzyme; a detergent additive and detergent compositioncontaining the enzyme preparation; as well as use of the enzymepreparation e.g. in the pulp and paper industry, the textile industry,the juice industry, for beer brewing, for animal feed and for bakingpurposes.

BACKGROUND OF THE INVENTION

Enzymes have been used for a long time for a variety of industrialapplications. For instance the use of enzymes in detergents, bothlaundry and dishwashing detergents, has become increasingly popular inrecent years. Further important uses of enzymes are in papermaking pulpprocessing, in the baking industry for improving the properties offlour, in the wine and juice industries for the degradation ofβ-glucans, in the textile industry for bio-polishing of cellulosicfabrics such as viscose, i.e. for obtaining a soft and smooth fabric bysubjecting the cellulosic fabrics to treatment by hemicellulolyticenzymes during their manufacture, and in animal feed for improving thedigestibility of vegetable protein sources.

It is, however, far from easy to obtain an optimal enzyme performancee.g. in a detergent system, as the detergent formulation and washingconditions (for instance high pH, high ionic strength, and the inclusionof certain surfactants and builders) may have a crucial impact on thestability and activity of the enzyme.

Since washing conditions are quite often alkaline, some enzymes at leastmight be expected to show an improved performance if the pI of theenzymes is shifted to a value approximating that of the pH duringapplication.

Similar considerations may apply to the use of enzymatic processes inother industries, e.g. one or more of the industries mentioned above.

E.g. when processing papermaking pulps, the lignocellulosic fibers maybe subjected to enzymatic hydrolysis. Hydrolysing enzymes for fibremodification may be lipase for hydrolysis of triglycerides in pitchdeposits, proteases for breakdown of structural proteins (e.g.extensin), and hemi-cellulase and pectinases for degradation of thecarbohydrate material constituting the fibre wall.

It is well established that the effect e.g. of carbohydrases is limiteddue to electrostatic repulsion. So far no economical or technicallyfeasible method for overcoming this limiting electrostatic repulsion hasbeen suggested. In WO 93/11296 and WO 93/07332 it is described how therepulsion can be reduced by enzymatic removal of negatively chargedglucoronic acid in the fibre matrix or by exchanging the counter ions onthe acid groups in the fibre. These procedures are, however, very costlysince bulk mass of lignocellulosic fibers must be treated with expensivespecialty enzymes or metal salts. The latter may also cause problems inthe internal water treatment of lignocellulosic fibre processinginstallations.

Furthermore, up till now it has been believed that the size of theenzyme molecules is another determining parameter for the effect ofenzymes acting on lignocellulosic fibers. Average fibre pore sizes havebeen claimed to be of the same magnitude as the average diameter of thesingle enzyme molecules (Viikari, L., Kantelinen, A., Ratto, M. &Sundquist, J. (1991), Enzymes in Biomass Conversion, Chpt.2: Enzymes inPulp and Paper Processing, p. 14, (Leatham, G. F. & Himmel, M. E.,eds.). Thus, it is still an unsolved problem how to improve the effectof enzymatic hydrolysis of lignocellulosic fibers.

DESCRIPTION OF THE INVENTION

Surprisingly, it has been found that when lipases, amylases,oxidoreductases, pectinases and/or hemicellulases are derivatised in away that masks the negatively charged side groups, this may lead tounexpected high increases in enzyme activity and/or in substrateavailability. This is despite the fact that such a derivatisationincreases the size of the enzyme molecules.

Thus, the electrostatic repulsion may be reduced through modification ofthe enzyme molecules in stead of modifying the substrate. Calculated inmass quantity, the amount of substrate is typically at least 100 timesmore than the mass of the enzyme product used in enzymatic processes,e.g. for treatment of the lignocellulosic fibers. Accordingly, it ismuch more economical to modify the enzyme instead of the lignocellulosicfibers.

The present invention relates to an enzyme preparation comprising amodified enzyme selected from the group consisting of amylases, lipases,oxidoreductases, pectinases and hemicellulases, said modified enzymehaving an improved performance due to an alkaline pI and/or an increasedsurface activity obtained by chemical modification or amino acidsubstitution.

It is obvious that the enzyme preparation of the invention may containone or more modified enzymes selected from the group consisting ofamylases, lipases, oxidoreductases, pectinases and hemicellulases eitheralone or in combination with other enzymes which have not been subjectedto a chemical modification or an amino acid substitution with thepurpose of obtaining an alkaline pI and/or an increased surfaceactivity.

In the present context, the term "improved performance" is intended toindicate that the modified enzyme, when subjected to the same standardtest conditions as the parent enzyme, exhibits an improved effectcompared to the parent enzyme. For enzymes intended to be included indetergent compositions, the modified enzyme is tested under standardwashing conditions and its performance, e.g. with respect to removingstains and soiling, is compared to that of the non-modified parentenzyme. The wash performance of the modified enzyme may not only beevaluated under laundry conditions, but also under dishwashingconditions. For enzymes intended to be used in papermaking pulpprocessing, the performance of the modified enzyme on unbleached oroxygen bleached kraft pulp is evaluated from the amount of lignin thatis dissolved from the pulp under a treatment with said endo-xylanase,subtracted the amount of lignin that is dissolved in a controltreatment, where addition of endo-xylanase is omitted. The dissolvedlignin is measured as the absorbance at 280 nanometers (Chpt. 5.1.4.2.by Lin, S. Y. in "Methods in Lignin Chemistry", Springer-Verlag, 1992),see example 6 below. A supplementary second parameter for measuring theeffect of a treatment of kraft pulp with e.g. an endo-xylanase (ahemicellulase) is the content of residual lignin in the pulp. The bestmeasure of the residual lignin content in the pulp is the kappa no.according to TAPPI procedure T236.

The isoelectric point, pI, is defined as the pH value at which theenzyme molecule is neutral, i.e. the sum of electrostatic charges (netelectrostatic charge) is equal to zero. In this sum of courseconsideration of the positive or negative nature of the individualelectrostatic charges must be taken into account. The pI mayconveniently be determined experimentally by isoelectric focusing or bytitrating a solution containing the enzyme.

The term "alkaline pI" is intended to indicate that the isoelectricpoint of the modified enzyme, as determined by isoelectric focusingunder standard conditions, is higher than 7.5. According to theinvention, it is generally preferred that the pI of the modified enzymeis at least 8.0, more preferably at least 8.5, most preferably at least9.0. According to the invention, the pI of the modified enzyme shouldpreferably be at least one pI unit, more preferably at least two pIunits, most preferably at least three pI units, higher than that of theparent enzyme.

The parent lipase may suitably be a microbial lipase. As such, theparent lipase may be selected from yeast, e.g. Candida; lipases,bacterial, e.g. Pseudomonas or Bacillus, lipases; or fungal, e.g.Humicola or Rhizomucor, lipases. More specifically, suitable lipases maybe the Rhizomucor miehei lipase (e.g. prepared as described in EP 238023), Thermomyces lanuginosa lipase e.g. prepared as described in EP 305216 (available from Novo Nordisk under the trade name Lipolase™),Humicola insolens lipase, Pseudomonas stutzeri lipase, Pseudomonascepacia lipase, Candida antarctica lipase A or B, or lipases from rGPL,Absidia blakesleena, Absidia corymbifera, Fusarium solani, Fusariumoxysporum, Penicillum cyclopium, Penicillum crustosum, Penicillumexpansum, Rhodotorula glutinis, Thiarosporella phaseolina, Rhizopusmicrosporus, Sporobolomyces shibatanus, Aureobasidium pullulans,Hansenula anomala, Geotricum penicillatum, Lactobacillus curvatus,Brochothrix thermosohata, Coprinus cinerius, Trichoderma harzanium,Trichoderma reesei, Rhizopus japonicus or Pseudomonas plantari. Otherexamples of suitable lipases may be variants of any one of the lipasesmentioned above, e.g. as described in WO 92/05249 or WO 93/11254.

In a preferred embodiment of the invention, the degree of residuallipase activity is preferably above about 15%.

Examples of suitable amylases include Bacillus amylases, e.g. Bacillusstearothermophilus amylase, Bacillus amyloliquefaciens amylase, Bacillussubtilis amylase or Bacillus licheniformis amylase (e.g. as availablefrom Novo Nordisk under the trade name Termamyl®), or Aspergillusamylases, e.g. Aspergillus niger or Aspergillus oryzae amylase. Otherexamples of suitable amylases may be variants of any one of the amylasesmentioned above, e.g. as described in U.S. Pat. No. 5,093,257, EP 252666, WO 91/00353, FR 2,676,456, EP 285 123, EP 525 610, PCT/DK93/00230.

The term "hemicellulase" is intended to include glycanases (apart fromcellulose- and starch-degrading enzymes), mannanases, galactomannases,xylanases, arabinanases, polyglucuronases or polygalacturonases.

Examples of suitable xylanases include Humicola insolens (see e.g. WO92/17573), Bacillus pumilus (see e.g. WO 92/03540), Bacillusstearathermophilus (see e.g. WO 91/18976, WO 91/10724), Bacillus sp.AC13 (see e.g. WO 94/01532), the genus Thermotoga (see e.g. WO93/19171), the genus Rhodothermus (see e.g. WO 93/08275), the genusDictyoglomus (see e.g. WO 92/18612), Tricoderma longibrachiatum andChainia sp. (see e.g. EP 0 353 342 A1), Thermoascus aurantiacus (seee.g. U.S. Pat. No. 4,966,850), Trichoderma harzianum and Trichodermareseei (see e.g. U.S. Pat. No. 4,725,544), Aureobasidium pullulans (seee.g. EP 0 373 107 A2), Thermomyces lanuginosus (see e.g. EP 0 456 033A2), Bacillus circulans (WO 91/18978), Aspergillus oryzae (see e.g. SU4610007), Thermomonospora fusca (see e.g. EP 0 473 545 A2), the genusStreptomyces (see e.g. U.S. Pat. No. 5,116,746), Streptomyces lividans(see e.g. WO 93/03155), Streptomyces viridosporus (see e.g. EP 496 671A1), Bacillus licheniformis (see e.g. JP 9213868) and Trichodermalongibrachiatum see W.J.J. van den Tweel et al.(Eds.), "Stability ofEnzymes",Proceedings of an International Symposium heeld in Maastricht,The Netherlands, 22-25 Nov. 1992, Fisk, R. S. and Simpson, pp.323-328!.Other examples of suitable xylanases may be variants of any one of thexylanases mentioned above

The term "oxidoreductases" is intended to include oxidases, laccases andperoxidases.

Examples of oxidoreductases include e.g. horseradish peroxidase, soybeanperoxidase or a peroxidase derived from Coprinus, e.g. C.cinereus, orderived from Bacillus, e.g. B. pumilus. Other examples include ligninperoxidases and mangan peroxidases e.g. from Phanerochaetechrysosporium. Further examples include laccases from Trametes, e.g. T.versicolor or T. villosa, and laccases from Polyporus pinsetus orPyricularia oryzae.

The term "pectinases" is intended to include polygalacturonases(EC3.2.1.15), pectinesterases (EC3.2.1.11), pectin lyases (EC4.2.2.10)and hemicellulases such as endo-1,3-β-xylosidase (EC 3.2.1.32), xylan1,4-β-xylosidase (EC 3.2.1.37) and α-L-arabinofuranosidase (EC3.2.1.55). A suitable source organism for pectinases may be Aspergillusniger.

It is to be understood that any of the enzymes mentioned in the presentspecification and claims may be produced by a given microorganism or,alternatively, may be a single (recombinant) enzyme, i.e. a componentessentially free of other enzymes or enzyme activity usually occurringin an enzyme product produced by a given microorganism, the singleenzyme being a recombinant enzyme, i.e. produced by cloning of a DNAsequence encoding the single enzyme and subsequent cell transformed withthe DNA sequence and expressed in a host. The host is preferably aheterologous host, but the host may under certain conditions also be thehomologous host.

The term "cellulosic fabric" is intended to include fabric originatingfrom xylan-containing cellulose fibers, e.g. from wood pulp. Examples ofcellulosic fabrics are viscose (rayon); Tencel®; all blends of viscosewith other fabrics such as viscose/polyester blends, viscose/cottonblends, viscose/wool blends; flax (linen) and ramie and other fabricsbased on xylan-containing cellulose fibers, including all blends ofcellulosic fabrics with other fabrics such as cotton, wool, andpolyester, e.g. viscose/polyester blends, viscose/cotton blends,viscose/wool blends, viscose/cotton/polyester blends, flax/cotton blendsetc.

In a preferred embodiment of the enzyme preparation of the invention,the enzyme is chemically modified by coupling an amine ligand to thecarboxyl group of glutamic acid or aspartic acid residues in the enzyme.By this chemical modification, the carboxylic acid groups areneutralized, thereby increasing the pI of the enzyme. The amine ligandis preferably an aminated sugar, aminated alcohol or aminatedpolyalcohol. Examples of suitable aminated sugars are glucosamine,isomeric forms thereof with the general formula C₆ H₁₃ O₅ N, oroligomers and polymers of the general formula C₆ H₁₁ O₄ N!_(n), forexample polymers of glucosamines such as chitosans. Oligomers andpolymers may be either branched or linear.

If an aminated alcohol is used for coupling to the carboxyl group, itshould generally contain at least 3 carbon atoms. Examples of suitableaminated alcohols are aminopropanol or aminobutanol. More preferably theamine ligand is an aminated polyalcohol. Polyalcohols should generallycontain at least 3 carbon atoms, and may for instance contain 6 carbonatoms. Examples of suitable aminated polyalcohols are glucamine,isomeric forms thereof with the general formula C₆ H₁₅ O₅ N, oroligomers and polymers thereof with the general formula C₆ H₁₃ O₄N!_(n), wherein n>1.

Other suitable amine ligands are amine substituted alkanes andderivatives thereof. Preferred examples of amine substituted alkanes andtheir derivatives are amino acids such as lysine, polylysine; esters ofamino acids; spermine; spermidine; putrescine; and the like.

The amine ligand such as an aminated sugar, alkane, alcohol orpolyalcohol and polymer thereof, should have at least one amino groupper monomeric unit, but should not be considered to be limited to havingonly one amino group per monomeric unit.

According to a preferred method, the coupling of the amine to thecarboxyl group of glutamic acid or aspartic acid residues is mediated bya crosslinking agent capable of binding a carboxyl group and an aminogroup. The coupling reaction may suitably be carried out by standardmethods as described by S. S. Wong, Chemistry of Protein Conjugation andCross-Linking, CRC Press, Boca Raton, Fla., USA, 1991, in particularChapter 2, IV, C, Chapter 4, IV and Chapter 5, II; or Wong and Wong,Enzyme Microb.Technol. 14, Nov. 1992, pp. 866-873. A particularlypreferred crosslinking agent for the coupling reaction is acarbodiimide, e.g. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

Methods of conjugating proteins with ligands using EDC can beimplemented according to manufacturer's description (e.g. PierceInstructions 0475 C, 22980 X; 22981 X; EDC) using either the protocolfor "Use of EDC for coupling of Haptens/small ligands to carrierProteins" or "Protocol for Efficient Two-Step coupling of Proteins inSolution Using EDC and N-hydroxysuccinimide orsulfo-N-hydroxysucciminide".

For example the enzyme may be dissolved, or transferred by dialysis ordesalting by size exclusion chromatography in a coupling buffer, suchas, for instance 50 mM MES pH 5.0 containing 200 mM sodium chloride. Theligand, e.g. glucosamine, may be dissolved in coupling buffer as well.The conjugation reaction may proceed by mixing enzyme and ligand to afinal concentration of 3 mg/ml for both enzyme and ligand followed bymixing with 5 mg of EDC per mg of enzyme. The conjugation reaction thenruns for 2 hours at room temperature with continuous stirring. Thereaction is terminated by removal of surplus reagent either by desaltingby size exclusion chromatography or by extensive dialysis, e.g. against0.2M ammonium acetate pH 6.9 at 5° C. The resulting derivative may thenbe stored at 5° C.

The degree of modification or incorporation of ligands may, of course,be controlled by adjustments in the initial enzyme, ligand and/orcarbodiimide concentration. Variations in pH or temperature of thecoupling buffer may also be included to optimise the conjugationreaction for a specific enzyme.

Naturally active site protection by substrate, substrate analogues andreversible inhibitors may be used to control of the modificationreaction.

In another preferred embodiment of the enzyme preparation of theinvention, the enzyme may be modified by substitution of one or moreamino acids. The invention therefore further relates to an enzymepreparation comprising a modified enzyme selected from the groupconsisting of an amylase, lipase, oxidoreductase, pectinase orhemicellulase, wherein at least one negatively charged or neutral aminoacid residue is substituted by a positively charged amino acid residueor, if the amino acid residue to be substituted is a negatively chargedamino acid, a neutral amino acid residue. The object of saidsubstitution is to provide a modified enzyme with an increased positivenet charge relative to the parent enzyme, as a higher positive netcharge results in a more alkaline pI.

When the parent enzyme is a lipase, the modified enzyme may be preparedby the general method described in WO 92/05249 (as well as describedbelow). In describing a modified lipase according to the invention, thefollowing nomenclature is used for ease of reference, using theconventional one-letter code for amino acid residues:

Original amino acid(s):position(s):substituted amino acid(s)

According to this nomenclature, for instance the substitution ofarginine for aspartic acid in position 165 is shown as: D165R

In one embodiment of a modified T. lanuginosus lipase, the electrostaticcharge of the enzyme may be changed by substituting one or more aminoacid residues located on the surface of the enzyme, in particular in oneor more of the positions 5, 43, 45, 50, 69, 70, 72, 94, 102, 105, 165,167, 199, 200 or 244, in combination with one or more of position 56,87, 96, 210 or 254. More specifically, one or more amino acid residuesmay be substituted as follows:

D5R

E43Q

E45Q

T50K

L69R

D70R

T72K

N94K

D102K

S105K

D165R

D167R/K

T199K

N200R

T244K

In combination with

E56K/R

E87K/R;N/Q

D96K/R

E210K/R

D254K/R

In a preferred embodiment the combination is/T72K/T244K/D102K/S105K/E87K/D96K/N94K/D165R/D167K/E43Q/E45Q/T50K/L69R/D70R/.

In one embodiment of a modified B. licheniformis amylase, theelectrostatic charge of the enzyme may be changed by substituting one ormore amino acid residues located on the surface of the enzyme, inparticular in one or more of the positions 53, 113, 114, 271, 419, 421or 458. More specifically, one or more amino acid residues may besubstituted as follows:

D53R/K

E113R/K

D114R/K

E271R/K

V419R/K

N421R/K

E447R/K

E458R/K

H471R/K

In one embodiment of a modified T. lanuginosus xylanase, theelectrostatic charge of the enzyme may be changed by substituting one ormore amino acid residues located on the surface of the enzyme, inparticular in one or more of the positions 7, 11, 30, 95, 110, 127, 155,156, 177, 181 or 183. More specifically, one or more amino acid residuesmay be substituted as follows:

E7R/K

E7T/S

E7Q/N

D11Q/N

D11R/K

E30R/K

N95R/K

D110R/K/S

D127K/R

N155R/K

A156R/K

Q177R/K

E181S/T

D183N/Q

D183R/K

In a further preferred embodiment of the enzyme preparation of theinvention, the enzyme may be modified by substitution of at least oneamino acid residue by at least one other amino acid residue to form anamino acid sequence specifying a glycosylation site recognized by amicroorganism capable of glycosylating enzymes, such as a fungus oryeast. The object of introducing glycosylation site(s) is to provide amodified enzyme with an increased positive net charge or an increasedhydrophilicity compared to that of the parent enzyme.

Preparation of modified enzymes by amino acid substitution

Several methods for introducing mutations into genes are known in theart. After a brief discussion of cloning enzyme-encoding DNA sequences,methods for generating mutations at specific sites within theenzyme-encoding sequence will be discussed.

Cloning a DNA sequence encoding an enzyme

The DNA sequence encoding a parent enzyme may be isolated from any cellor microorganism producing the enzyme in question by various methods,well known in the art. First a genomic DNA and/or cDNA library should beconstructed using chromosomal DNA or messenger RNA from the organismthat produces the enzyme to be studied. Then, if the amino acid sequenceof the enzyme is known, homologous, labelled oligonucleotide probes maybe synthesized and used to identify enzyme-encoding clones from agenomic library prepared from the organism in question. Alternatively, alabelled oligonucleotide probe containing sequences homologous to aknown enzyme could be used as a probe to identify enzyme-encodingclones, using hybridization and washing conditions of lower stringency.

Yet another method for identifying enzyme-encoding clones would involveinserting fragments of genomic DNA into an expression vector, such as aplasmid, transforming enzyme-negative bacteria with the resultinggenomic DNA library, and then plating the transformed bacteria onto agarcontaining a substrate for enzyme thereby allowing clones expressing theenzyme to be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g. the phosphoamiditemethod described by S. L. Beaucage and M. H. Caruthers, TetrahedronLetters 22, 1981, pp. 1859-1869, or the method described by Matthes etal., The EMBO J. 3, 1984, pp. 801-805. According to the phosphoamiditemethod, oligonucleotides are synthesized, e.g. in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in appropriatevectors.

Finally, the DNA sequence may be of mixed genomic and synthetic, mixedsynthetic and cDNA or mixed genomic and cDNA origin prepared by ligatingfragments of synthetic, genomic or cDNA origin (as appropriate), thefragments corresponding to various parts of the entire DNA sequence, inaccordance with standard techniques. The DNA sequence may also beprepared by polymerase chain reaction (PCR) using specific primers, forinstance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et al.,Science 239, 1988, pp. 487-491.

Site-directed mutagenesis

Once an enzyme-encoding DNA sequence has been isolated, and desirablesites for mutation identified, mutations may be introduced usingsynthetic oligonucleotides. These oligonucleotides contain nucleotidesequences flanking the desired mutation sites; mutant nucleotides areinserted during oligonucleotide synthesis. In a specific method, asingle-stranded gap of DNA, bridging the enzyme-encoding sequence, iscreated in a vector carrying the enzyme gene. Then the syntheticnucleotide, bearing the desired mutation, is annealed to a homologousportion of the single-stranded DNA. The remaining gap is then filled inwith DNA polymerase I (Klenow fragment) and the construct is ligatedusing T4 ligase. A specific example of this method is described inMorinaga et al., (1984, Biotechnology 2:646-639). U.S. Pat. No.4,760,025, by Estell et al., issued Jul. 26, 1988, discloses theintroduction of oligonucleotides encoding multiple mutations byperforming minor alterations of the cassette, however, an even greatervariety of mutations can be introduced at any one time by the Morinagamethod, because a multitude of oligonucleotides, of various lengths, canbe introduced.

Another method of introducing mutations into enzyme-encoding DNAsequences is described in Nelson and Long, Analytical Biochemistry 180,1989, pp. 147-151. It involves the 3-step generation of a PCR fragmentcontaining the desired mutation introduced by using a chemicallysynthesized DNA strand as one of the primers in the PCR reactions. Fromthe PCR-generated fragment, a DNA fragment carrying the mutation may beisolated by cleavage with restriction endonucleases and reinserted intoan expression plasmid.

Expression of modified enzymes

According to the invention, a mutated enzyme-encoding DNA sequenceproduced by methods described above, or any alternative methods known inthe art, can be expressed, in enzyme form, using an expression vectorwhich typically includes control sequences encoding a promoter,operator, ribosome binding site, translation initiation signal, and,optionally, a repressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding amodified enzyme of the invention encoding may be any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e. a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding the enzyme variant of theinvention, especially in a bacterial host, are the promoter of the lacoperon of E.coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis α-amylase gene(amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amym), the promoters of the Bacillus Amyloliquefaciensα-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylBgenes etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralα-amylase, A. niger acid stable α-amylase, A. niger glucoamylase,Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triosephosphate isomerase or A. nidulans acetamidase.

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the enzyme variant ofthe invention. Termination and polyadenylation sequences may suitably bederived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B.subtilis or B.licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amds, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g. as described in WO 91/17243.

The procedures used to ligate the DNA construct of the inventionencoding an enzyme variant, the promoter, terminator and other elements,respectively, and to insert them into suitable vectors containing theinformation necessary for replication, are well known to persons skilledin the art (cf., for instance, Sambrook et al. Molecular Cloning; ALaboratory Manual, CSH, NY, 1989).

The cell of the invention either comprising a DNA construct or anexpression vector as defined above is advantageously used as a host cellin the recombinant production of an enzyme variant of the invention. Thecell may be transformed with the DNA construct encoding the modifiedenzyme, conveniently by integrating the DNA construct in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g. by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described below in connection with thedifferent types of host cells.

The host cell may be a cell of a higher organism such as a mammal or aninsect, but is preferably a microbial cell, e.g. a bacterial or a fungal(including yeast) cell.

Examples of suitable bacteria are gram-positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gram-negative bacteria such asE.coli. The transformation of the bacteria may for instance be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

The yeast organism may favourably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. Thefilamentous fungus may advantageously belong to a species ofAspergillus, e.g. Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

The modified enzyme may be produced by cultivating a host cell asdescribed above under conditions conducive to the production of themodified enzyme and recovering the modified enzyme from the cells and/orculture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the modified enzyme of the invention. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedrecipes (e.g. in catalogues of the American Type Culture Collection).

The modified enzyme secreted from the host cells may conveniently berecovered from the culture medium by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulphate, followed by chromatographic proceduressuch as ion exchange chromatography, affinity chromatography, or thelike.

Detergent Additives and Compositions

Due to their improved washing and/or dishwashing performance, modifiedamylases or lipases of the invention are particularly well suited forinclusion into detergent compositions, e.g. detergent compositionsintended for performance in the range of pH 7-13, particularly the rangeof pH 8-11.

According to the invention, the modified amylase or lipase may be addedas a component of a detergent composition. As such, it may be includedin the detergent composition in the form of a detergent additive. Thedetergent composition as well as the detergent additive may additionallycomprise one or more other enzymes conventionally used in detergents,such as proteases, oxidases, peroxidases and cellulases.

In a specific aspect, the invention provides a detergent additive. Themodified amylase or lipase and optionally one or more other enzymes maybe included in a detergent composition by adding separate additivescontaining one or more enzymes, or by adding a combined additivecomprising all of these enzymes. A detergent additive of the invention,i.e. a separated additive or a combined additive, can be formulated e.g.as granulates, liquids, slurries, etc. Preferred detergent additiveformulations are granulates, in particular non-dusting granulates,liquids, in particular stabilized liquids, slurries, or protectedenzymes.

Non-dusting granulates may be produced, e.g. as disclosed in U.S. Pat.No. 4,106,991 and U.S. Pat. No. 4,661,452, and may optionally be coatedby methods known in the art. The detergent enzymes may be mixed beforeor after granulation.

Liquid enzyme preparations may, for instance, be stabilized by adding apolyol such as propylene glycol, a sugar or sugar alcohol, lactic acidor boric acid according to established methods. Other enzyme stabilizersare well known in the art. Protected enzymes may be prepared accordingto the method disclosed in EP 238 216.

In a still further aspect, the invention relates to a detergentcomposition comprising a modified amylase or lipase of the invention.

The detergent composition of the invention may be in any convenientform, e.g. as powder, granules or liquid. A liquid detergent may beaqueous, typically containing up to 90% of water and 0-20% of organicsolvent, or non-aqueous, e.g. as described in EP Patent 120,659.

The detergent composition comprises a surfactant which may be anionic,non-ionic, cationic, amphoteric or a mixture of these types. Thedetergent will usually contain 0-50% of anionic surfactant such aslinear alkylbenzene sulfonate, alpha-olefinsulfonate, alkyl sulfate,alcohol ethoxy sulfate or soap. It may also contain 0-40% of non-ionicsurfactant such as nonyl phenol ethoxylate or alcohol ethoxylate.Furthermore, it may contain an N-(polyhydroxyalkyl)-fatty acid amidesurfactant (e.g. as described in WO 92/06154).

The detergent may contain 1-40% of detergent builders such as zeolite,di or triphosphate, phosphonate, citrate, NTA, EDTA or DTPA, alkenylsuccinic anhydride, or silicate, or it may be unbuilt (i.e. essentiallyfree of a detergent builder).

The detergent composition of the invention may be stabilized usingconventional stabilizing agents for the enzyme(s), e.g. a polyol such ase.g. propylene glycol, a sugar or sugar alcohol, lactic acid, boricacid, or a boric acid derivative, e.g. an aromatic borate ester, and thecomposition may be formulated as described in e.g. WO 92/19709 and WO92/19708. Other enzyme stabilizers are well known in the art.

The detergent composition of the invention may contain bleaching agents,e.g. perborate, percarbonate and/or activator, tetraacetyl ethylenediamine, or nonanoyloxybenzene sulfonate, and may be formulated asdescribed in e.g. WO 92/07057.

The detergent composition of the invention may also contain otherconventional detergent ingredients, e.g. deflocculating polymers, fabricconditioners, foam boosters, foam depressors, anti-corrosion agents,soil-suspending agents, sequestering agents, anti-soil redepositionagents, dyes, bactericides, optical brighteners and perfumes as well asenzymes as mentioned above.

Particular forms of detergent composition within the scope of theinvention and containing a modified amylase or lipase of the inventioninclude:

a) A detergent composition formulated as a detergent powder containingphosphate builder, anionic surfactant, nonionic surfactant, silicate,alkali to adjust to desired pH in use, and neutral inorganic salt.

b) A detergent composition formulated as a detergent powder containingzeolite builder, anionic surfactant, nonionic surfactant, acrylic orequivalent polymer, silicate, alkali to adjust to desired pH in use, andneutral inorganic salt.

c) A detergent composition formulated as an aqueous detergent liquidcomprising anionic surfactant, nonionic surfactant, organic acid,alkali, with a pH in use adjusted to a value between 7 and 11.

d) A detergent composition formulated as a nonaqueous detergent liquidcomprising a liquid nonionic surfactant consisting essentially of linearalkoxylated primary alcohol, phosphate builder, alkali, with a pH in useadjusted to a value between about 7 and 11.

e) A compact detergent composition formulated as a detergent powder inthe form of a granulate having a bulk density of at least 600 g/l,containing anionic surfactant and nonionic surf actant, phosphatebuilder, sodium silicate, and little or substantially no neutralinorganic salt.

f) A compact detergent composition formulated as a detergent powder inthe form of a granulate having a bulk density of at least 600 g/l,containing anionic surfactant and nonionic surfactant, zeolite builder,sodium silicate, and little or substantially no neutral inorganic salt.

g) A detergent composition formulated as a detergent powder containinganionic surfactant, nonionic surfactant, acrylic polymer, fatty acidsoap, sodium carbonate, sodium sulfate, clay particles, and sodiumsilicate.

h) A liquid compact detergent comprising 5-65% by weight of surfactant,0-50% by weight of builder and 0-30% by weight of electrolyte.

i) A compact granular detergent comprising linear alkyl benzenesulphonate, tallow alkyl sulphate, C45 alkyl sulphate, C₄₋₅ alcohol 7times ethoxylated, tallow alcohol 11 times ethoxylated, dispersant,silicone fluid, trisodium citrate, citric acid, zeolite, maleic acidacrylic acid copolymer, DETMPA, cellulase, protease, lipase, anamylolytic enzyme, sodium silicate, sodium sulphate, PVP, perborate andaccelerator.

j) A granular detergent comprising sodium linear C₁₋₂ alkyl benzenesulfonate, sodium sulfate, zeolite A, sodium nitrilotriacetate,cellulase, PVP, TAED, boric acid, perborate and accelerator.

k) A liquid detergent comprising C₁₂₋₁₄ alkenyl succinic acid, citricacid monohydrate, sodium C₁₂₋₁₅ alkyl sulphate, sodium sulfate of C₁₂₋₁₅alcohol 2 times ethoxylated, C₁₂₋₁₅ alcohol 7 times ethoxylated, C₁₂₋₁₅alcohol 5 times ethoxylated, diethylene triamine penta (methylenephosphonic acid), oleic acid, ethanol, propanediol, protease, cellulase,PVP, suds supressor, NaOH, perborate and accelerator.

Furthermore, examples of suitable detergent compositions in which amodified amylase or lipase of the invention may advantageously beincluded comprises the detergent compositions described in EP 373 850,EP 378 261, WO 92/19709, EP 381 397, EP 486 073, WO 92/19707, EP 407225, and WO 92/13054.

Dishwashing Compositions

Apart from a modified amylase or lipase of the invention, thedishwashing detergent composition comprises a surfactant which may beanionic, non-ionic, cationic, amphoteric or a mixture of these types.The detergent will contain 0-90% of non-ionic surfactant such as low- tonon-foaming ethoxylated propoxylated straight-chain alcohols.

The detergent composition may contain detergent builder salts ofinorganic and/or organic types. The detergent builders may be subdividedinto phosphorus-containing and non-phosphorus-containing types. Thedetergent composition usually contains 1-90% of detergent builders.

Examples of phosphorus-containing inorganic alkaline detergent builders,when present, include the water-soluble salts especially alkali metalpyrophosphates, orthophosphates, polyphosphates, and phosphonates.Examples of non-phosphorus-containing inorganic builders, when present,include water-soluble alkali metal carbonates, borates and silicates aswell as the various types of water-insoluble crystalline or amorphousalumino silicates of which zeolites are the bestknown representatives.

Examples of suitable organic builders include the alkali metal, ammoniumand substituted ammonium, citrates, succinates, malonates, fatty acidsulphonates, carboxymetoxy succinates, ammonium polyacetates,carboxylates, polycarboxylates, aminopolycarboxylates, polyacetylcarboxylates and polyhydroxsulphonates.

Other suitable organic builders include the higher molecular weightpolymers and co-polymers known to have builder properties, for exampleappropriate polyacrylic acid, polymaleic and polyacrylic/polymaleic acidcopolymers and their salts.

The dishwashing detergent composition may contain bleaching agents ofthe chlorine/bromine-type or the oxygen-type. Examples of inorganicchlorine/bromine-type bleaches are lithium, sodium or calciumhypochlorite and hypobromite as well as chlorinated trisodium phosphate.Examples of organic chlorine/bromine-type bleaches are heterocyclicN-bromo and N-chloro imides such as trichloroisocyanuric,tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids,and salts thereof with water-solubilizing cations such as potassium andsodium. Hydantoin compounds are also suitable.

The oxygen bleaches are preferred, for example in the form of aninorganic persalt, preferably with a bleach precursor or as a peroxyacid compound. Typical examples of suitable peroxy bleach compounds arealkali metal perborates, both tetrahydrates and monohydrates, alkalimetal percarbonates, persilicates and perphosphates. Preferred activatormaterials are TAED and glycerol triacetate.

The dishwashing detergent composition of the invention may be stabilizedusing conventional stabilizing agents for the enzyme(s), e.g. a polyolsuch as e.g.propylene glycol, a sugar or a sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g. an aromatic borate ester.

The dishwashing detergent composition of the invention may also containother conventional detergent ingredients, e.g. deflocculant material,filler material, foam depressors, anti-corrosion agents, soil-suspendingagents, sequestering agents, anti-soil redeposition agents, dehydratingagents, yes, bactericides, fluorescers, thickeners and perfumes.

Other applications

It is contemplated that, dependent on the specificity of the enzyme, itmay be employed for one or possibly more of the applications mentionedabove, i.e. in the baking industry, in the wine and juice industry, foranimal feed, and in papermaking pulp processing. In a particularembodiment, the enzyme preparation of the invention may comprise acombination of one or more enzymes selected from the group consisting ofmodified amylase, lipase and hemicellulase with one or more otherenzymes.

Pulp and paper applications

In the papermaking pulp industry, the enzyme preparation according tothe invention may be applied advantageously e.g. as follows:

For debarking, i.e. pretreatment with hydrolytic enzymes such aspectolytic and/or hemi-cellulolytic enzymes may degrade the pectin-richcambium layer prior to debarking in mechanical drums resulting inadvantageous energy savings.

For defibration (refining or beating), i.e. treatment of materialcontaining cellulosic fibers with hydrolytic enzymes such as pectolyticand/or hemi-cellulolytic enzymes prior to the refining or beating whichresults in reduction of the energy consumption due to the hydrolysingeffect of the enzymes on the surfaces of the fibers. Use of the enzymepreparation according to the present invention may result in higherenergy savings as compared to use of unmodified enzymes, since themodified enzyme(s) possess a higher ability to penetrate fibre walls.

For fibre modification, i.e. improvement of fibre properties wherepartial hydrolysis across the fibre wall is needed which requires deeperpenetrating enzymes (e.g. in order to make coarse fibers more flexible).Deep treatment of fibers has so far not been possible for high yieldpulps e.g. mechanical pulps or mixtures of recycled pulps. Thisrestriction has been ascribed to the nature of the fibre wall structurethat prevents the passage of enzyme molecules due to physicalrestriction of the pore matrix of the fibre wall. Surprisingly, themodified (i.e. derivatised) enzymes of the enzyme preparation of theinvention are more capable of penetrating into the fibre wall. Thisfinding indicates that also for high yield pulps, the negatively chargedacid groups on the micro fibrillar surfaces restrict the penetration ofenzymes molecules by electrostatic repulsion.

For drainage: The drainability of papermaking pulps may be improved bytreatment of the pulp with hydrolysing enzymes such as e.g.hemi-cellulases, lipase and/or pectinases. Use of the enzyme preparationaccording to the invention may be more effective, e.g. result in ahigher degree of loosening bundles of strongly hydrated micro-fibrils inthe fines fraction that limits the rate of drainage by blocking hollowspaces between the fibers and in the wire mesh of the paper machine.

For inter fibre bonding. Hydrolytic enzymes are applied in themanufacture of pulps for improving the inter fibre bonding. The enzymesrinse the fibre surfaces for noncellulosic impurities and fines, thuscreating larger area of exposed cellulose and hemi-cellulose whichimproves the fibre-to-fibre hydrogen binding capacity. This process isalso referred to as dehornification. Paper and board produced with ahemi-cellulase containing enzyme preparation according to the inventionmay have an improved strength or a reduced grammage, a smoother surfaceand an improved printability. This improvement is due to the improvedpenetrability of the modified/derivatised enzyme(s).

For enzymatic deinking. Partial hydrolysis of recycled paper uponpulping by use of hydrolysing enzymes such as e.g. lipase, pectinases,and hemi-cellulases are known to facilitate the removal andagglomeration of ink particles. Use of an enzyme preparation accordingto the invention may give a more effective loosening of ink from thesurface structure due to a better penetration of the enzyme moleculesinto the fibrillar matrix of the fibre wall, thus softening the surfacewhereby ink particles are effectively loosened.

For bleaching of kraft pulp, see example 6 below. Treatment of oxygenbleached kraft pulp with endo-xylanase (a hemicellulase) is performedindustrially for lowering the content of residual lignin in the pulp,thus reducing the need for chemicals in subsequent bleaching. Tominimise the need for addition of mineral acid and to avoid problemswith corrosion, it is required that the treatment with endoxylanase isperformed at a high pH. However, partly due to electrostatic repulsionthe performance of the majority of commercial endo-xylanase products isreduced as the pH is increased above 7.

The treatment of lignocellulosic pulp may, e.g., be performed asdescribed in WO 93/08275, WO 91/02839 and WO 92/03608.

Textile applications

In another embodiment, the present invention relates to use of theenzyme preparation according to the present invention in thebio-polishing process. Bio-Polishing is a specific treatment of the yarnsurface which improves fabric quality with respect to handle andappearance without loss of fabric wettability. The most importanteffects of Bio-Polishing can be characterized by less fuzz and pilling,increased gloss/luster, improved fabric handle, increased durablesoftness and altered water absorbency. Bio-Polishing usually takes placein the wet processing of the manufacture of knitted and woven fabrics.Wet processing comprises such steps as e.g. desizing, scouring,bleaching, washing, dying/printing and finishing. During each of thesesteps, the fabric is more or less subjected to mechanical action. Ingeneral, after the textiles have been knitted or woven, the fabricproceeds to a desizing stage, followed by a scouring stage, etc.Desizing is the act of removing size from textiles. Prior to weaving onmechanical looms, warp yarns are often coated with size starch or starchderivatives in order to increase their tensile strength. After weaving,the size coating must be removed before further processing the fabric inorder to ensure a homogeneous and wash-proof result. The preferredmethod of desizing is enzymatic hydrolysis of the size by the action ofamylases. It is known that in order to achieve the effects ofBio-Polishing, a combination of enzymatic action and mechanical actionis required. It is also known that if the enzymatic treatment iscombined with a conventional treatment with softening agents,"super-softness" is achievable. It is contemplated that use of theenzyme preparation of the invention comprising amylase for enzymaticdesizing is advantageous; and that use of the enzyme preparation of theinvention comprising xylanase for bio-polishing of cellulosic fabrics,especially for viscose or Tencel® or blends thereof with other fabricsas mentioned above, is advantageous. Bio-polishing may be obtained byapplying the method described e.g. in WO 93/20278.

Baking

In yet another embodiment, the present invention relates to use of theenzyme preparation according to the present invention, especially achemically modified xylanase, amylase, lipase, laccase and/or oxidasepreparation, in baking flour so as to improve the development,elasticity and/or stability of dough and/or the volume, crumb structureand/or anti-staling properties of the baked product. Although the enzymepreparation may be used for the preparation of dough or baked productsprepared from any type of flour or meal (e.g. based on rye, barley, oat,or maize), the enzyme preparation of the invention have been found to beparticularly useful in the preparation of dough or baked products madefrom wheat or comprising substantial amounts of wheat. The bakedproducts produced with an enzyme preparation of the invention includesbread, rolls, baquettes and the like. For baking purposes the enzymepreparation of the invention may be used as having e.g. xylanase,lipase, amylase, oxidase or laccase as the only or major enzymaticactivity, or may be used in combination with other enzymes such as alipase, an amylase, an oxidase (e.g. glucose oxidase, peroxidase), alaccase and/or a protease; the lipase, amylase, oxidase and laccaseoptionally being modified as described herein.

Beer brewing

In yet another embodiment, the present invention relates to use of anenzyme preparation according to the invention in the beer brewingindustry in particular to improve the filterability of wort e.g.containing barley and/or sorghum malt. The xylanase and/or amylasepreparation may be used in the same manner as pentosanasesconventionally used for brewing, e.g. as described by Vietor et al.,1993, J. Inst. Brew. , May-June, 99, pp. 243-248, and EP 227 159.Furthermore, the xylanase preparation may be used for treatment ofbrewers spent grain, i.e. residuals from beer wort production containingbarley or malted barley or other cereals, so as to improve theutilization of the residuals for, e.g., animal feed.

Juice etc.

In yet another embodiment, the present invention relates to use of anenzyme preparation according to the invention in the juice industry.

It is contemplated that the enzyme preparation of the invention, i.e. axylanase preparation, is useful in the preparation of fruit or vegetablejuice in order to increase yield, and in the enzymatic hydrolysis ofvarious plant cell wall-derived materials or waste materials, e.g. frompaper production, or agricultural residues such as wheat-straw, corncobs, whole corn plants, nut shells, grass, vegetable hulls, bean hulls,spent grains, sugar beet pulp, and the like. The plant material may bedegraded in order to improve different kinds of processing, facilitatepurification or extraction of other component than the xylans likepurification of beta-glucan or beta-glucan oligomers from cereals,improve the feed value, decrease the water binding capacity, improve thedegradability in waste water plants, improve the conversion of e.g.grass and corn to ensilage, etc.

Finally, a xylanase preparation of the invention may be used inmodifying the viscosity of plant cell wall derived material. Forinstance, the xylanases may be used to reduce the viscosity of feedcontaining xylan, to promote processing of viscous xylan containingmaterial as in wheat separation, and to reduce viscosity in the brewingprocess.

Animal feed

In yet another embodiment, the present invention relates to use of anenzyme preparation according to the invention in animal feed (or for thetreatment of animal feed prior to ingestion by the animal). The enzymepreparation is preferably added to the feed in an amount which isefficient for improving the digestibility of vegetable protein sources,e.g. cereals and legumes. Thus, e.g. a xylanase preparation of thepresent invention may be used for modification of animal feed and mayexert its effect either in vitro (by modifying components of the feed)or in vivo. The xylanase preparation is particularly suited for additionto animal feed compositions containing high amounts of arabinoxylans andglucuronoxylans, e.g. feed containing cereals such as barley, wheat, ryeor oats or maize. When added to feed the xylanase significantly improvesthe in vivo break-down of plant cell wall material partly due to areduction of the intestinal viscosity (Bedford et al., Proceedings ofthe 1st symposium on Enzymes in Animal Nutrition, 1993, pp. 73-77),whereby a better utilization of the plant nutrients by the animal isachieved. Thereby, the growth rate and/or feed conversion ratio (i.e.the weight of ingested feed relative to weight gain) of the animal isimproved.

The following examples further illustrate the present invention, andthey are not intended to be in any way limiting to the scope of theinvention as claimed.

EXAMPLE 1

Expression of Humicola lanuginosa lipase in Aspergillus oryzae:

Cloning of Humicola lanuginosa lipase and expression andcharacterization of the lipase in Aspergillus oryzae is described in EPapplication 305,216. The expression plasmid used is named p960.

The expression plasmid used in this application is identical to p960,except for minor modifications just 3' to the lipase coding region. Themodifications were made the following way: p960 was digested with NruIand BamHI restriction enzymes. Between these two sites the BamHI/NheIfragment from plasmid pBR322, in which the NheI fragment was filled inwith Klenow polymerase, was cloned, thereby creating plasmid pAO1 (FIG.3), which contains unique BamHI and NheI sites. Between these uniquesites BamHI/XbaI fragments from p960 was cloned to give pAHL (FIG. 4).

Site-directed in vitro mutagenisation of lipase gene:

The approach used for introducing mutations into the lipase gene isdescribed in Nelson & Long, Analytical Biochemistry, 180, 147-151(1989). It involves the 3-step generation of a PCR (polymerase chainreaction) fragment containing the desired mutation introduced by using achemically synthesized DNA-strand as one of the primers in thePCR-reactions. From the PCR generated fragment, a DNA fragment carryingthe mutation can be isolated by cleavage with restriction enzymes andre-inserted into the expression plasmid. This method is thoroughlydescribed below. In FIGS. 5 and 6 the method is further outlined.

Construction of a plasmid expressing the D165R/D167K variant of Humicolalanuginosa lipase

Linearization of plasmid pAHL:

The circular plasmid pAHL was linearized with the restriction enzymeSphI in the following 50 μl reaction mixture: 50 mM NaCl, 10 mMTris-HCl, pH 7.9, 10 MM MgCl₂, 1 mM dithiothreitol, 1 μg plasmid and 2units of SphI. The digestion was carried out for 2 hours at 37° C. Thereaction mixture was extracted with phenol (equilibrated with Tris-HCl,pH 7.5) and precipitated by adding 2 volumes of ice-cold 96% ethanol.After centrifugation and drying of the pellet, the linearized DNA wasdissolved in 50 μl H2O and the concentration estimated on an agarosegel.

3-step PCR mutagenesis:

As shown in FIG. 6, 3-step mutagenisation involves the use of fourprimers:

    ______________________________________    Mutagenisation primer (=A):    5'-CCATATGAAAACACTTTGATTCTATACCCATTTCC-3'    PCR Helper 1 (=B):    5'-GGTCATCCAGTCACTGAGACCCTCTACCTATTAAATCGGC-3'    PCR Helper 2 (=C):    5'-CCATGGCTTTCACGGTGTCT-3'    PCR Handle (=D):    5'-GGTCATCCAGTCACTGAGAC-3'    ______________________________________

Helper 1 and helper 2 are complementary to sequences outside the codingregion, and can thus be used in combination with any mutagenisationprimer in the construction of a mutant sequence.

All 3 steps were carried out in the following buffer containing: 10 mMTris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, 0.2 mM DATP,0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM TTP, 2.5 units Taq polymerase.

In step 1, 100 pmol primer A, 100 pmol primer B and 1 fmol linearizedplasmid was added to a total of 100 μl reaction mixture and 15 cyclesconsisting of 2 minutes at 95° C., 2 minutes at 37° C. and 3 minutes at72° C. were carried out.

The concentration of the PCR product was estimated on an agarose gel.Then, step 2 was carried out. 0.6 pmol step 1 product and 1 fmollinearized plasmid was contained in a total of 100 μl of the previouslymentioned buffer and 1 cycle consisting of 5 minutes at 95° C., 2minutes at 37° C. and 10 minutes at 72° C. was carried out.

To the step 2 reaction mixture, 100 pmol primer C and 100 pmol primer Dis added (1 μl of each) and 20 cycles consisting of 2 minutes at 95° C.,2 minutes at 37° C. and 3 minutes at 72° C. were carried out. Thismanipulation comprised step 3 in the mutagenisation procedure.

Isolation of mutated restriction fragment:

The product from step 3 was isolated from an agarose gel andre-dissolved in 20 μl H₂ O. Then, it was digested with the restrictionenzymes BamHI and BstXI in a total volume of 50 μl with the followingcomposition: 100 mM NaCl, 50 mM Tris-HCl, pH 7.9, 10 mM MgCl₂, 1 mM DTT,10 units of BamHI and 10 units of BstXI. Incubation was at 37° C. for 2hours. The 733 bp BamHI/BstXI fragment was isolated from an agarose gel.

Ligation to expression vector pAHL:

The expression plasmid pAHL was cleaved with BamHI and BstXI underconditions indicated above and the large fragment was isolated from anagarose gel. To this vector, the mutated fragment isolated above wasligated and the ligation mix was used to transform E.coli. The presenceand orientation of the fragment was verified by cleavage of a plasmidpreparation from a transformant with restriction enzymes. Sequenceanalysis was carried out on the double-stranded plasmid using theDyeDeoxy™ Terminater Cycle Sequencing Kit (Applied Biosystems) on an ABIDNA sequencer, model 373A. The plasmid was named pAHLD165R/D167K and isidentical to pAHL, except for the substituted codons.

EXAMPLE 2

Primers used for the construction of other Humicola lipase variants.

The following mutations were incorporated using the same method asdescribed in example 1. The primers used for the modifications arelisted below.

    ______________________________________    Mutations             Primer A sequence    ______________________________________    E87K/D96K/             5'-    D102K    GCCGGAGCAAATCTTATTTATTTCTTTCAACTTG             AAGTTAAG-             ATTCCCGATCCAGTTTTTTATGGAACGAGA-3'    E210K    5'-GCTGTAACCGAACTTGCGCGGCGGGAG-3'    T199K/N200R             5'-AGGGACAATATCCCTCTTGTGGGTAATGCG-3'    ______________________________________

EXAMPLE 3

Construction of plasmids expressing combination variants of Humicolalipase.

The plasmids pAHLD165R/D167K/D102K/D96K/E87K,

pAHLD165R/D167K/D102K/D96K/E87K/E210K

pAHLD165R/D167K/D102K/D96K/E87K/T199K/N200R

pAHLD165R/D167K/D102K/D96K/E87K/T199K/N200R/E210K

were constructed by performing successive mutagenisation steps using theappropriate primers.

EXAMPLE 4

Expression of lipase variants in Aspergillus Transformation ofAspergillus oryzae (general procedure).

100 ml of YPD (Sherman et al., Methods in Yeast Genetics, Cold SpringHarbor Laboratory, 1981) was inoculated with spores of A. oryzae andincubated with shaking for about 24 hours. The mycelium was harvested byfiltration through miracloth and washed with 200 ml of 0.6M MgSO₄. Themycelium was suspended in 15 ml of 1.2M MgSO₄, 10 mM NaH₂ PO₄, pH=5.8.The suspension was cooled on ice and 1 ml of buffer containing 120 mg ofNovozym® 234, batch 1687 was added. After 5 min., 1 ml of 12 mg/ml BSA(Sigma type H25) was added and incubation with gentle agitationcontinued for 1.5-2.5 hours at 37° C. until a large number ofprotoplasts was visible in a sample inspected under the microscope.

The suspension was filtered through miracloth, the filtrate transferredto a sterile tube and overlayed with 5 ml of 0.6M sorbitol, 100 mMTris-HCl, pH=7.0. Centrifugation was performed for 15 min. at 1000 g andthe protoplasts were collected from the top of the MgSO₄ cushion. 2volumes of STC (1.2M sorbitol, 10 mM Tris-HCl, pH=7.5, 10 mM CaCl₂) wereadded to the protoplast suspension and the mixture was centrifugated for5 min. at 1000 g. The protoplast pellet was resuspended in 3 ml of STCand repelleted. This was repeated. Finally, the protoplasts wereresuspended in 0.2-1 ml of STC. 100 μl of protoplast suspension wasmixed with 5-25 μg of p3SR2 (an A. nidulans amds gene carrying plasmiddescribed in Hynes et al., Mol. and Cel. Biol., Vol. 3, No. 8,1430-1439, Aug. 1983) in 10 μl of STC. The mixture was left at roomtemperature for 25 min. 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl₂and 10 mM Tris-HCl, pH=7.5 was added and carefully mixed (twice) andfinally 0.85 ml of the same solution was added and carefully mixed. Themixture was left at room temperature for 25 min., spun at 2.500 g for 15min. and the pellet was resuspended in 2 ml of 1.2M sorbitol. After onemore sedimentation the protoplasts were spread on minimal plates (Cove,Biochem. Biophys. Acta 113 (1966) 51-56) containing 1.0M sucrose,pH=7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibitbackground growth. After incubation for 4-7 days at 37° C. spores werepicked, suspended in sterile water and spread for single colonies. Thisprocedure was repeated and spores of a single colony after the secondreisolation were stored as a defined transformant.

EXAMPLE 5

Expression of lipase variants in A. oryzae

The plasmids described above were transformed into A. oryzaeIFO 4177 bycotransformation with p3SR2 containing the amds gene from A. nidulans asdescribed in the above example. Protoplasts prepared as described wereincubated with a mixture of equal amounts of expression plasmid andp3SR2, approximately 5 μg of each were used. Transformants which coulduse acetamide as sole nitrogen source were reisolated twice. Aftergrowth on YPD for three days, culture supernatants were analyzed usingan assay for lipase activity. The best transformant was selected forfurther studies and grown in a 1 l shake-flask on 200 ml FG4 medium (3%soy meal, 3% maltodextrin, 1% peptone, pH adjusted to 7.0 with 4M NaOH)for 4 days at 30° C.

EXAMPLE 6

pI values of Lipolase and variants thereof

The theoretical pI values of Lipolase and modifications thereof havebeen determined based on the sequence numbers of titratable groups inthe residues Asp (D), Glu (E), Lys(K), Arg(R), His(H) and Tyr (Y) withinthe pH range 1-14.

The pI values are listed below:

    ______________________________________    Lipase wild-type (Lipolase)  pI 4.7    +D165R/D167K/D102K/D96K/EB7K pI 7.6    +D165R/D167K/D102K/D96K/E87K/E210K                                 pI 8.1    +D165R/D167K/D102K/D96K/E87K/T199K/N200R                                 pI 8.2    +D165R/D167K/D102K/D96K/E87K/T199K/N200R/E210K                                 pI 8.5    ______________________________________

EXAMPLE 7A

Conjugation of Lipolase™ with glucosamine, polylysine and polyarginine,respectively, mediated by EDC

Conjugation of Lipolase™ (a lipase expressed in and produced byAspergillus oryzae; produced and sold by Novo Nordisk A/S, Bagsvaerd,Denmark) with glucosamine, polylysine and polyarginine, respectively,through carbodiimide mediated coupling was performed according tostandard procedures.

An enzyme stock solution was prepared by dissolving approximately 50mg/ml of highly purified Lipolase™ in 50 mM boric acid/NaOH at pH 9.0.The enzyme was diluted in coupling buffer (50 mM MES pH 5.0 containing200 mM sodium chloride). The glucosamine, polylysine (MW 8200) andpolyarginine (MW 6000), respectively, was dissolved in coupling bufferas well.

The conjugation reaction proceeded by mixing enzyme andpolylysine/polyarginine to a final concentration of 3 mg/ml for bothenzyme and glucosamine followed by addition to 5 mg of EDC per mg ofenzyme to mediate the reaction. The conjugation reaction continued for 2hours at room temperature with continuos magnetic stirring.

The reaction was terminated by removing surplus reagent by extensivedialysis against 0.1M ammonium hydrogencarbonate pH 8 at 5° C. (forpolylysine and polyarginine) and agains 0.2M ammonium acetate pH 6.9 at5° C. (for glucosamine). The derivative was stored at 5° C.

The prepared Lipolase™-glucosamine derivative has a pI value of 9.5 asdetermined by isolectric focusing and 21.7% residual Lipolase™ activitywhen compared to wild-type Lipolase™.The prepared Lipolase™-polylysinederivative has a pI value of 9.5 as determined by isolectric focusingand 47.9% residual Lipolase™ activity when compared to wild-typeLipolase™. The prepared Lipolase™-polyarginine derivative has a pI valueof 9.5 as determined by isolectric focusing and 27.1% residual Lipolase™activity when compared to wild-type Lipolase™.

The activity was measured according to the standard Novo NordiskLipolase method AF-95-GB (available from Novo Nordisk A/S on request)which is hereby incorporated by reference.

EXAMPLE 7B

Conjugation of Termamyl® with glucosamine mediated by EDC

Conjugation of Termamyl® (an amylase expressed in and produced by astrain of Bacillus licheniformis, produced and sold by Novo Nordisk A/S,Bagsvaerd, Denmark) with glucosamine through carbodiimide mediatedcoupling was performed according to standard procedures.

An enzyme stock solution was prepared by dissolving approximately 50mg/ml of highly purified Termamyl™ in Britton-Robinson buffer at pH 9(0.04M phosphoric acid, acetic acid, boric acid; adjustment of pH bytitration with 0.2N NaOH). The enzyme was diluted in coupling buffer (50mM MES pH 5.0 containing 200 mM sodium chloride). The glucosamine wasdissolved in coupling buffer as well.

The conjugation reaction proceeded by mixing enzyme and glucosamine to afinal concentration of 3 mg/ml for the enzyme and 3, 0.6 and 0,3 mg/mlof glucosamine followed by addition to 5 mg of EDC per mg ofglucosamine. The conjugation reactions continued for 2 hours at roomtemperature with continuous magnetic stirring.

The reaction was terminated by removing surplus reagent by extensivedialysis towards 0.2M ammonium acetate pH 6.9 at 5° C. The derivativeswere stored at 5° C.

The amylase activity is determined by the standard Novo Nordisk methodAF-124-GB (available from Novo Nordisk A/S on request) for determinationof amylase activity in Termamyl® preparations. The method is herebyincorporated by reference.

The following variants were prepared:

    ______________________________________                   Glucos-   Amylase    TERMAMYL       amino     activity    pI    ______________________________________    Deriva-           3 mg/ml     3 mg/ml    3.90 KNU/ml                                           9.5    tive #1    Deriva-           3 mg/ml     3 mg/ml    8.80 KNU/ml                                           9-9.5    tive #2    Deriva-           3 mg/ml     3 mg/ml   15.40 KNU/ml                                           8.5-9    tive #3    ______________________________________

EXAMPLE 7C

Conjugation of Termamyl® with glucosamine mediated by EDC at pH 6

Conjugation of Termamyl® (an amylase expressed in and produced by astrain of Bacillus licheniformis) with the glucosamine throughcarbodiimide mediated coupling is performed according to standardprocedures.

An enzyme stock solution was prepared by dissolving approximately 50mg/ml of highly purified Termamyl® in Britton-Robinson buffer at pH 9(0,04M phosphoric acid, acetic acid, boric acid; adjustment of pH bytitration with 0.2N NaOH). The enzyme was diluted in coupling buffer (50mM MES pH 6.0 containing 200 mM sodium chloride). The glucosamine wasdissolved in coupling buffer as well.

The conjugation reaction proceeded by mixing enzyme and glucosamine to afinal concentration of 3 mg/ml for the enzyme and of 3 and 1,5 mg/ml ofglucosamine followed by addition to 5 mg of EDC per mg ofglucosamination. The conjugation reactions continued for 2 hours at roomtemperature with continuous magnetic stirring.

The reaction was terminated by removing surplus reagent by extensivedialysis towards 0.2M ammonium acetate pH 6.9 at 5° C. The derivativeswere stored at 5° C.

The amylase activity is determined by the standard Novo Nordisk methodAF-124-GB (available from Novo Nordisk A/S on request) for determinationof amylase activity in Termamyl® preparations. The method is herebyincorporated by reference.

The following variants were prepared:

    ______________________________________                   Glucos-   Amylase    TERMAMYL       amino     activity   pI    ______________________________________    Deriva-           3 mg/ml       3 mg/ml 10.1 KNU/ml                                          8-9    tive #5    Deriva-           3 mg/ml     1.5 mg/ml 15.3 KNU/ml                                          7.5-8.5    tive #6    ______________________________________

EXAMPLE 7D

Conjugation of Thermomyces lanuginosus xylanase with glucosaminemediated by EDC

Conjugation of Thermomyces lanuginosus xylanase with glucosamine throughcarbodiimide mediated coupling was performed according to standardprocedures.

An enzyme stock solution was prepared by dialysis against couplingbuffer for equilibration. The enzyme was diluted in coupling buffer (50mM MES pH 5.0 containing 200 mM sodium chloride). The glucosamine wasdissolved in coupling buffer as well.

The conjugation reaction proceeded by mixing enzyme and glucosamine to afinal concentration of 2 mg/ml for both enzyme and glucosamine followedby addition to 5 mg of EDC per mg of enzyme to mediate the reaction. Theconjugation reaction continued for 2 hours at room temperature withcontinuos magnetic stirring.

The reaction was terminated by removing surplus reagent by extensivedialysis against Britton-Robinson buffer (see Example 1B) at pH 7 at 5°C. The derivative was stored at 5° C.

The xylanase-glucosamine derivative prepared according to the abovedescribed procedure was shown to be monomeric by size-exclusionchromatography on a TSK-G2000SW column, has a pI value of 9 asdetermined by isolectric focusing and 3.8% residual xylanase activitywhen compared to wild-type T. lanuginosus xylanase. The activity ismeasured according to the standard Novo Nordisk xylanase methodAF-293.9/1-GB (available from Novo Nordisk A/S on request) which ishereby incorporated by reference.

EXAMPLE 8

Interfacial activity of Lipolase and the glucosamine derivative thereof

By means of tensiometry it was shown that glucosamination of Lipolase™results in a significant increase in the interfacial activity of theenzyme at alkaline pH values.

The measurements were performed with a Sigma 70 tensiometer from KSV,Finland, equipped with a Wilhelmy Pt-plate. The experiments were carriedout by injecting 25 μl highly purified enzyme (adjusted to OD₂₈₀ nm=1.66 for both Lipolase™ and the glucosamine derivative ("Lipolase-GA")into a 100 ml buffer-solution, while following the surface tension γwith time.

Measurements were performed at 25° C. in 50 mM Tris pH 7+500 mM NaCl and50 mM glycine pH10+500 mM NaCl. Addition of an excess of neutral saltwas done in order to increase the adsorption of enzyme at the air-waterinterface.

Already at pH 7 it appears that the extent of adsorption is increased bythe glucosamination (FIG. 1). Going from pH 7 to pH 10 does not in anymajor way alter the adsorption of native Lipolase™. On the other hand,the pI of the enzyme derivative resulted in a significant increase inits surface activity (FIG. 2).

EXAMPLE 9

Improved lipolytic performance in the presence of alcohol ethoxylates

Using a monolayer equipment (KSV-5000, KSV Instruments, Finland) it wasdemonstrated that glucosamination of Lipolase™ considerably increasedthe lipolytic action of this lipase in the presence of long-chainalcohol ethoxylates. A large number of non-ionic surfactants present inmost currently used detergents are alcohol ethoxylates (e.g. Dobanol25-7).

Experimental

A mixed monolayer of a well-defined overall composition, made up of adiglyceride substrate and a monocomponent alcohol ethoxylate was spreadon an aqueous subphase. The surface pressure was adjusted to the desiredvalue, and a well-defined amount of lipase was injected into thesubphase. Lipolytic action is manifested through the speed of a mobilebarrier compressing the monolayer in order to maintain constant surfacepressure as insoluble substrate molecules are hydrolysed into more watersoluble reaction products. Using this assay, lipases are discriminatedby:

B: The final area-fraction of substrate left unhydrolysed by the lipase.

The table below illustrates that the glucosamine and polylysinederivatives of Lipolase™, respectively, performs considerably better inthe presence of alcohol ethoxylates. In addition it is demonstrated thatthe performance of lipase in the presence of alcohol ethoxylates isincreased when the net charge of the lipase is increased.

Improved tolerance of Lipolase™ towards alcohol ethoxylates uponglucosamination

    ______________________________________    Enzyme              β    ______________________________________    Lipolase™ (wild-type)                        59%    Wild-type + E210R   56%    Wild-type + T199K/N200R                        53%    Wild-type + D102K/S105K                        49%    Glucosaminated derivative                        0%    Polylysine derivative                        0%    ______________________________________     Note: 10 mM glycine buffer, substrate: Dicaprin, 10 Lipase Units (LU), pH     10.0, 25° C., 30 mN/m. Additive: Heptaethylene monooctadecyl ether

EXAMPLE 10

Washing performance of amylase and glucosamine derivative thereof

Fabric with coloured starch

In order to visualize the detergency of the enzyme preparation of theinvention, coloured starch was produced according to the followingprocedure. 50 g of potato starch was solubilized in 500 ml H₂ O andheated to 80° C. Then 5 g of the dye Cibacron Blue 3GA was addedtogether with 100 g sodium sulphate and 500 ml deionized water. Themixture was heated for 15 minutes. Subsequently 5 g trisodiumphosphatewas added, the temperature was lowered to 50° C. and the mixture wasagitated for 75 minutes and then cooled to room temperature. Acentrifuging step was applied to remove surplus of unreacted dye.

100% cotton fabric was then submerged into the solution, pressed througha roller and line dryed. The remission of the fabric at 660 nm was thenmeasured and should be in the range of 35-45.

Washing procedure

Swatches of the dyed fabric were washed in glass beakers with agitationby a magnetic stirrer.

Volume: 60 ml

Wash time: 20 min

Rinse: 15 min

Swatches: 6 swatches with a diameter of 2.5 cm

Temperature: 55° C.

Detergent: Commercial high-pH European automatic dishwashing detergent,3 g/l

pH: 10.2

Drying: Line drying

Repetitions: 1

Enzymes

Termamyl™

Glucosaminated Termamyl® (TRMEDC1), see Example 7B

Evaluation

The starch removal on the swatches are measured in respect to remissionat 660 nm, once on both sides. (Apparatus: Elrepho from DataColor/Switzerland).

Data R and standard deviations ().

    ______________________________________    Enzyme   0 KNU/l   0.4 KNU/l 1.0 KNU/l                                         3.0 KNU/l    ______________________________________    Termamyl 53.65     68.55     76.40   82.86             (1.04)    (0.34)    (0.59)  (0.12)    TRMEDC1  53.65     73.50     82.33   86.20             (1.04)    (0.58)    (0.24)  (0.15)    ______________________________________

The results demonstrate that the glucosaminated enzyme performssignificantly better than than non-modified Termamyl®.

EXAMPLE 11

Improved performance of Lipolase™ in the presence of detergents

High pI derivatives of Lipolase™ were prepared as described in example7A. Dervatisation of Lipolase™ was carried out at a ratio of Lipolase topolylysine/polyarginine of 1:1, based on weight.

EDC3: Lipolase™ conjugated with glucosamine

EDC10: Lipolase™ conjugated with poly-L-arginine (6.0 kDa, Sigma P4663)

EDC11: Lipolase™ conjugated with poly-L-lysine (8.2 kDa, Sigma P6516)

The high pI of the derivatives were confirmed by IEF. Samples wereloaded onto an Ampholine PAG-plate pH 3.5-9.5 (Pharmacia) and runaccording to the manufacturer's instructions. The three conjugates wereshown to have a pI above 9.5.

The residual activities of the conjugates were measured using thestandard Novo Nordisk Lipolase™ method AF-95-GB which is available fromNovo Nordisk A/S upon request.

The residual activities of the Lipolase derivatives are shown in thetable below:

    ______________________________________                                      Residual    Enzyme    A.sub.280                     LU/ml     LU/A.sub.208                                      activity (%)    ______________________________________    Lipolase  1.0    4077      4077   100    EDC3      1.61   1300      813    20    EDC10     0.41   727       1773   43    EDC11     0.57   811       1423   35    ______________________________________

The performance of the Lipolase conjugates in the presence of detergentswere investigated in an assay using paranitrophenyl palmitate(pNP-palmitate, Sigma N2752) as a substrate. The absorbance of thep-nitrophenol released upon lipase catalused hydrolysis was measured at405 nm as a function of time. The assay was run in 0.1M Tris-HCl, 0.352mM CaCl₂, pH 10, containing either Dobanol 25-7 (nonionic detergent) orAriel Ultra (full laundry detergent, commercial available from thecompany Procter & Gamble; enzymatic activities removed by heating).Lipolase and derivatives thereof were dosed on an activity bases (3.75LU/ml).

The results are shown in the table below.

The results demonstrate that the high pI conjugates have considerablehigher activity.

The improvement factor IF in the table, defined as

    IF=(ΔA.sub.405 derivative)/(ΔA.sub.405 wild-type)

after 30 minutes, expresses the amount of lipase variant protein neededto obtain the same effect as that obtained with the reference wild-typelipase.

    ______________________________________                                IF       IF                                Dobanol 25-7                                         Ariel Ultra    Enzyme  Description                      Dosage    (4 μg/ml)                                         (100 μg/ml)    ______________________________________    Lipolase            Wild-type 3.75 LU/ml                                1.0      1.0    poly-Arg            WT + p-Arg                      2.1 μg/ml                                1.0      1.0            (control)    poly-Lys            WT + p-Lys                      2.6 μg/ml                                0.5      0.8            (control)    glucosamine            WT +      12.5 mM   0.7      n.a.            glucosamine    EDC3    glucosamine                      3.75 LU/ml                                6.2      n.a.            derivative    EDC10   poly-Arg  3.75 LU/ml                                4.5      2.7            derivative    EDC11   poly-Lys  3.75 LU/ml                                6.3      3.5            derivative    ______________________________________     n.a.: data not available

EXAMPLE 12

Washing performance of Lipolase and derivatives thereof of the invention

Swatches

Textile swatches containing fat with a dyestuff as an indicator for fatremoval were prepared as follows: Bleached cotton (NT 2116 from NordiskTekstil) was cut into pieces of 3.5*3.5 cm. 0.075% (w/w) of Sudan redwas added to lard at 70° C.; the mixture was kept at 5° C. and heated upto about 70° C. before use. 6 μl of the lard/Sudan red was applied tothe centre of each swatch. The swatches were incubated at 70° C. for 30minutes and kept overnight prior to the experiment. Two swatches wereused for each experiment.

Conditions

The swatches were washed in glass beakers with agitation by a magneticstirrer.

Volume: 100 ml

Detergent: European model detergent

Swatches: 6 swatches

pH: 10.2

Wash time: 20 min

Rinse: 15 min

Temperature: 30° C.

Drying: line drying

Repetitions: 3

Enzymes

Lipolase™

Lipolase™-glucosamine (EDC3), see example 7A and 11

Lipolase™-polylysine (EDC11), see example 7A and 11

Lipolase™-polyarginine (EDC10), see example 7A and 11

Evaluations

The detergency of the enzymes was evaluated by measuring the remissionat 460 nm (on a Elrepho-meter) on both sides of the swatches.

Delta R (remission) versus no enzyme.

    ______________________________________           300    750    1500      3000 10000           LU/l   LU/l   LU/l      LU/l LU/l    ______________________________________    Lipolase 3.5      4.8    5.6     5.5  7.0    EDC3     4.5      5.8    5.2     6.7  7.8    EDC10    4.1      6.1    7.3     7.9  11.6    EDC11    5.0      6.0    8.2     9.3  11.3    ______________________________________

Further, Lipolase™ and the derivatives EDC10 and EDC11 were tested in a3-cycle mini-wash assay under the following conditions:

Enzymes: 0, 300, 750, 1500, 3000, 10000 LU/1

Swatches/fabric: see above under swatches

Detergent: Heavy Duty Powder composition containing 1.17 g/l

Linear alkylbenzene sulphonate, 0.15 g/l AEO (Dobanol

25-7), 1.25 g/l sodium triphosphate, 1 g/l sodium sulphate, 0.45 g/l

sodium carbonate, 0.15 g/l sodium metasilicate; pH 10.2.

Wash: 6 swatches in 100 ml water per beaker were washed at 30° C. for 20minutes, rinsed for 15 minutes in running tap water and dryed overnightat room conditions.

Evaluation: After each wash cycle the reflectance was measured on bothsides of the swatches at 460 nm. The improvement factor IF wascalculated as described in example 11.

The following results were obtained:

    ______________________________________            Enzyme         IF    ______________________________________            Lipolase™   1.0            EDC10          1.5            EDC11          2.6    ______________________________________

EXAMPLE 13

Modified endo-xylanase (hemicellulase) for kraft pulp

Samples of oxygen bleached kraft pulp was repulped at 1.5% consistencyin a laboratory pulper with 10.000 revolutions according to SCAN C18 anddrained on a Buchner funnel. The pH was adjusted with sulfuric acid. Thesamples were diluted to 10% consistency. A purified endo-xylanasepreparation obtained from T.lanuginosus was added to two pulp sampleswith pH 7 and 8.5 respectively, at a rate of 765 U/kg. One U is definedas the amount of endo-xylanase that in one minute hydrolyses onemicromole of beta 1-4 linkages in a xylan polymer. To two other sampleswith pH 7 and 8.5 were added T. lanuginosus endo-xylanase modifiedaccording to the invention (see e.g. example 1D), also at a rate of 765U/kg. Finally two control samples were adjusted in pH to respectively 7and 8.5.

The 6 pulp samples were now incubated in closed plastic bags immersed inthermostated water at 60° C. The bags were kneaded by hand 30 secondsevery 15 minutes. After three hours incubation time the pulp sampleswere drained. Samples of the waterphase were filtered through a 45micrometer filter in order to remove any micro-fibrils from the pulp,and the final pH and the absorbance at 280 nm were determined. The pulpsamples were then washed with deionized water, and the level of residuallignin was measured as kappa no. The results are given in the tablebelow. In each figure the control value has been subtracted.

At pH 7, the effect of the treatment with the modified (derivatised)endo-xylanase is an increase of the amount of released lignin of factor2.9 and, correspondingly, a decrease in the residual lignin level whichis a reduction of the kappa number with a factor 2.9.

At the alkaline pH 8.5, the amount of released lignin is 460% higherwhen the modified endo-xylanase is used as compared to the unmodifiedreference enzyme. The decrease in kappa number is 12.6 times higher withthe modified endo-xylanase as compared to the reference (the unmodifiedenzyme).

                  TABLE    ______________________________________                      Released Decrease in           Final pH   lignin   kappa no.    ______________________________________    Reference             7.02         1.736    0.49    pH 7    Derivative             7.00         5.036    1.42    pH 7    Reference             8.36         0.854    0.08    pH 8.5    Derivative             8.37         3.890    1.01    pH 8.5    ______________________________________

The results show that the performance of an endo-xylanase is improveddrastically, also in the alkaline pH range, when modified, i.e.derivatised, according to the present invention.

THE DRAWINGS

The invention is further illustated by the drawings in which

FIG. 1 shows the adsorption of Lipolase and GA-lipolase at A/W-interfaceat pH 7, 25° C.;

FIG. 2 shows the adsorption of Lipolase and GA-lipolase at A/W-interfaceat pH 10, 25° C.;

FIG. 3 shows the plasmide paO1;

FIG. 4 shows the plasmide pAHL;

FIG. 5 and FIG. 6 illustrate the method of 3-step PCR mutagenesis.

We claim:
 1. An enzyme preparation comprising a modified enzyme, whereinthe modification comprises coupling an amine group to the carboxyl groupof a glutamic acid or aspartic acid residue, and wherein the enzyme isselected from the group consisting of an amylase, lipase,oxidoreductase, pectinase, and hemicellulase.
 2. An enzyme preparationaccording to claim 1, wherein the pI of the modified enzyme is as leastone pI unit higher than that of the parent enzyme.
 3. An enzymepreparation according to claim 1 wherein the amine is an aminated sugar,aminated alkane, aminated alcohol, aminated polyalcohol or amino acid oran ester or other derivatives thereof.
 4. An enzyme preparationaccording to claim 3, wherein the aminated sugar is glucosamine,isomeric forms thereof, or oligomers or polymers thereof.
 5. An enzymepreparation according to claim 3, wherein the aminated alcohol having atleast 3 carbon atoms, for instance aminopropanol or aminobutanol.
 6. Anenzyme preparation according to claim 3, wherein the aminatedpolyalcohol is D-glucamine, isomers thereof, or oligomers or polymersthereof.
 7. An enzyme preparation according to claim 3, wherein theamino acid is lysine, spermine, spermidine, putrescine, or polymersthereof such as polylysine and polyarginine.
 8. An enzyme preparationaccording to claim 1, wherein the coupling of the amine to the carboxylgroup of glutamic acid or aspartic acid residues is mediated by acrosslinking agent capable of binding a carboxyl group and an aminogroup.
 9. An enzyme preparation according to claim 8, wherein thecrosslinking agent is selected from the group consisting ofcarbodiimides, isoxazolium derivatives, chloroformates orcarbonyldiimidazole.
 10. An enzyme preparation according to claim 9,wherein the crosslinking agent is a carbodiimide.
 11. An enzymepreparation according to claim 1, wherein the pI of the modified enzymeis at least 8.0.
 12. An enzyme preparation according to claim 11,wherein the pI of the modified enzyme is at least 8.5.
 13. An enzymepreparation according to claim 12, wherein the pI of the modified enzymeis at least 9.0.
 14. An enzyme preparation according to claim 2 whereinthe pI of the modified enzyme is as least two pI units higher than thatof the parent enzyme.
 15. An enzyme preparation according to claim 14,wherein the pI of the modified enzyme is as least three pI units higherthan that of the parent enzyme.
 16. A detergent additive comprising anenzyme preparation according to claim 1 in the form of a non-dustinggranulate, stabilized liquid or protected enzyme.
 17. A detergentcomposition comprising an enzyme preparation according to claim 1 aswell as a surfactant.
 18. A detergent composition according to claim 17wherein the enzyme preparation is present in a concentrationcorresponding to 0.01-100, preferably 0.05-60, mg of enzyme protein perliter of wash liquor.
 19. A detergent composition according to claim 18which is a dishwashing detergent.
 20. A method for the treatment oflignocellulosic fibers, wherein the fibers are treated with an enzymepreparation according to claim 1, in an amount which is efficient forimproving the fiber properties.
 21. A method according to claim 20,wherein the enzyme preparation comprises a pectinase, a hemicellulase,an endo-xylanase, or a combination thereof.
 22. A method according toclaim 20 wherein the lignocellulosic fibers are kraft pulp which istreated with the enzyme preparation in an amount which is efficient forsubstantially lowering the content of residual ligning in the pulp. 23.A method according to claim 20 for enzymatic deinking of recycled paperpulp, wherein the enzyme preparation is applied in an amount which isefficient for effective deinking of the fibre surface.